Bone fractures and defects are common in the general population, and management of fractures is the leading cause of all trauma admission for adolescents and adults under 65 years, generating $1.2 billion in hospital costs (1). While fracture healing is efficient and typically results in newly formed bone, a number of adverse conditions impair the healing process, leading to delayed healing and nonunion in a small but significant number of patients. Orthopedic surgeons have known for years that smoking is a major contributor to a variety of bone conditions including osteoporosis, lumbar disc disease, healing of fractures, the rate of hip fractures and bone cancer (2). Recent studies have started revealing the pathological mechanisms of smoking on musculoskeletal injuries and established a real and reproducible relationship between smoking and musculoskeletal diseases (2, 3). In particular, clinical studies have found that smokers have a significant longer time to clinical union, and a higher incidence of delayed union compared with non-smokers (4, 5). Addressing the needs and providing optimal care for these patients have important social and economical impact.
Artificial bone grafts regenerated by donor cells and biomaterials, also known as tissue engineered bone constructs (TEBC), represent a novel approach that overcomes the donor limitation and increases the efficacy for defect repair and healing. TEBC requires both an osteogenic cell source and a substrate material that can support the regeneration of healthy bone. Human mesenchymal stem cells (hMSCs), which are known to be responsible for the normal turnover and maintenance of adult mesenchymal tissues in vivo, are inducible osteoprogenitor cells and have become cell of choice in bone tissue regeneration (6, 7). On the other hand, natural and synthetic scaffolds seeded with culture expanded hMSCs have been extensively studied in bone tissue repair and regeneration and shown promising clinical results (8, 9, 10). The combination of hMSC with bioactive scaffolds has become an attractive approach to enhance bone healing for the patients such as smokers and elder patients who have impaired regeneration capacity and not responsive to conventional therapeutic intervention.
The treatment of fractures in young patients and osteoporotic fractures in elderly patients is a major part of trauma care. For young adults, critical size defect and delayed union are the major problem related to morbidity during healing, whereas osteoporosis-related complications are the major problem to the ageing population. Although the risks of tobacco smoking have been well known for decades, there are more than 50 million smokers in the U.S. and over 500,000 deaths can be attributed to smoking (2, 16). Cigarette smoking contributes to musculoskeletal diseases and influences an array of orthopaedic conditions from bone mineral density to the rate of hip fractures and fracture healing (2, 3). Smoking is known to reduce blood supply, has high levels of reactive oxygen intermediates, and low concentration of oxidant vitamins. Although nicotine at low doses may be stimulatory, high dose nicotine is directly toxic to proliferating osteoblasts (17). Nicotine has been shown to inhibit the strength of repair in a fracture model and in distraction osteogenesis in rabbit (18, 19). In addition to nicotine, other components of cigarette smoke may also be harmful. In animal study, tobacco extract not containing nicotine significantly reduced the mechanical strength of healing femoral fractures in rats (20). Clinical studies have found that smokers have a significant longer time to clinical union, and a higher incidence of delayed union compared with non-smokers (4). Smokers also have a higher rate of nonunion and poorer results after fusion of the ankle and spine (21). Smoking also induces osteoporosis and leads to increased risk of fracture in elderly men (22, 23). Together, these results suggest that cigarette smoking, whether it is the nicotine or other components of cigarettes, is a significant contributing factor in bone diseases and fracture healing.
The pathological mechanism that results in the adverse effects of cigarette smoking on bone disease and healing has begun to be revealed. Smoking influences the biochemical interactions and cellular properties that occur during fracture healing which leads to an impaired healing. For example, TGF-β1 is essential for bone formation and osteogenic differentiation, and TGF-β1 knockout mice have defect in bone strength and structure (24). In a recent study, TGF-β1 serum concentrations, which are considered to be one of the most important markers of fracture healing, are reduced by smoking, and the reduction of TGF-β1 serum concentration in smokers is statistically significant during the 4th week after surgery (25). In addition to the molecular milieu, smoking also affects cellular properties that are crucial mediators of wound repair and healing. Mesenchymal stem cells (MSCs) regulate the normal bone homeostasis and are inducible osteoblast progenitors (6). MSCs are also significant source of cytokines that mediate inflammatory response and participate in wound healing. Smoking compromises
hMSC's ability for cytokine secretion and down-regulate their osteoblastic differentiation due to reduced blood supply and high concentration of free radicals and toxins associated with smoking, inhibiting patient's self-healing process (26, 27). Implantation of constructs containing hMSCs from healthy donor could augment self-healing capacity and improve clinical outcome.
Promotion of bone healing through biological means is a major therapeutic option for trauma surgeons. Bone graft is the second most commonly transplanted tissue following blood (28). While numerous types of grafts have been used, the ideal bone graft should be an optimal combination of osteogenic, osteoinductive, and osteoconductive properties. These porous implantable materials not only act as a 3D template for bone growth but their degradation products also have no toxic effects. To further enhance the bone regeneration potential, these bioresorbable scaffolds are often impregnated with suitable cell types that augment bone regeneration process. In this effort, it is advantageous to select a scaffolding material to mimic natural tissue composition in addition to promoting hMSC proliferation and differentiation. Chitosan, gelatin, and hydroxyapatite in various combinations are among frequently studied biomimetic composite scaffolds for bone regeneration because of their chemical similarity to natural extracellular matrix (ECM) (11, 29, 30, 31). Chitosan, a linear polysaccharide is composed of glucosamine and N-acetyl glucosamine units linked by β(1-4) glycosidic bonds. Structural similarity of chitosan with various glycosaminoglycans (GAGs) found in the extracellular matrix of bone and cartilage has made chitosan an attractive material in bone and cartilage tissue regeneration. The cationic nature of chitosan allows for mimicking the ECM-rich environment of bone tissue through the formation of insoluble ionic complexes with anionic molecules such as growth factors, glycosaminoglycans (GAG), and proteoglycans benefiting cell growth and tissue formation (32). Gelatin is a partially denaturalized collagen and retains moieties that facilitate cell adhesion and influence cell behaviors (33). The abundance of functional groups in gelatin allows for interaction with growth factors and forms a favorable microenvironment for tissue regeneration. Hydroxyapatite (HA) is the mineral component of natural bone ECM and has been used to improve biocompatibility and hard tissue integration through the sequestering of serum proteins (34). Our laboratory has developed a composite of hydroxyapatite-chitosan-gelatin and demonstrated that the presence of HA improves protein adsorption in the porous HCG scaffolds and enhances hMSC long-term growth and osteogenic differentiation upon induction (11).
Recent advances in human mesenchymal stem cells (hMSC) provide a promising cell source that is readily available from adult donor, is easy to be expanded in culture, and has high potential to differentiate into bone tissue. Originally isolated from bone marrow, but now identified in multiple tissue sources, MSCs are multi-potent progenitor cells responsible for the repair and regeneration of mesenchymal tissue such as bone, cartilage, fat, and muscle (6, 35). Along with considerable in vitro studies, autologous bone marrow-derived MSCs have also been used in various bone diseases and demonstrated their therapeutic potential in patients (36, 37, 38, 39). MSC have been combined with 3-D biomaterials to repair the site-specific bone defect with good results (40, 41). In both large and small animal models, the implantation of MSC-seeded constructs has demonstrated their ability to accelerate the repair of femoral defects, craniomaxillofacial deformities, and spinal fusion (42, 43). In addition to their multipotentiality, MSCs have unique immunosuppressive properties which allow allogeneic transplantation without the need of immunosuppression (44, 45). This has significant implication in human therapy because MSCs derived from healthy donors can be cryopreserved and made available for patients in a variety of acute and chronic clinical settings. Studies have shown that MSCs can survive freezing temperatures without significant change in viability, indicating their potential for future “off-the-shelf” therapeutic applications (46). Despite the success in cryopreserving cells in suspension, methods for the cryopreservation of the constructs loaded with cells has not been reported. Although the “off-the-shelf” constructs are cost effective and provide the flexibility needed for the surgical room, cryopreservation of TEBCs remains a technical barrier and little is known about the impact of such procedure on cell viability and regeneration potency (15).
Our laboratory has developed a hydroxyapatite-chitosan-gelatin (HCG) scaffold and successfully demonstrated its superior properties for bone regeneration when infused with hMSCs (11, 12). We have also developed a perfusion bioreactor system that integrates cell seeding and long-term tissue growth, which significantly improving system efficiency and construct properties (13, 14). Using the perfusion bioreactor system developed in our lab, we have also shown that dynamic cell seeding into the center of the 3D HCG porous scaffolds and supports long-term construct growth, thereby streamlining the fabrication process (see preliminary results). The in vitro and animal studies have shown promising results for HCG's application in bone regeneration. HCG scaffolds and the perfusion bioreactor system establishes a technology platform required for the fabrication of functional bone constructs from hMSC.